胡海燕， 徐 蕾， 戚菲菲， 王丹丹， 劉 靜， 曾 勝， 劉北星

(中國醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院 免疫學(xué)教研室，遼寧 沈陽 110122 )

天然輔助細(xì)胞在初次和再次RSV感染所誘發(fā)氣道炎癥反應(yīng)中的作用

胡海燕， 徐 蕾， 戚菲菲， 王丹丹， 劉 靜， 曾 勝， 劉北星*

(中國醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院 免疫學(xué)教研室，遼寧 沈陽 110122 )

天然輔助細(xì)胞(natural helper cell，NHC)是近期發(fā)現(xiàn)的二型固有淋巴細(xì)胞的一種，因其在病毒感染后分泌大量Th2型細(xì)胞因子，故在病毒感染所誘發(fā)的氣道炎癥反應(yīng)中發(fā)揮重要作用。呼吸道合胞病毒(Respiratory Syncytial Virus, RSV)是最常見的呼吸道病毒，也是一個(gè)反復(fù)感染的過程。NHC在RSV感染過程發(fā)揮的作用尤其是在再次感染中具體作用尚不清楚，故本研究建立初次及再次感染模型，通過計(jì)數(shù)肺泡灌洗液(BALF)中炎性細(xì)胞數(shù)量，HE染色觀察肺病理炎癥反應(yīng)，Real-time RT-PCR法檢測肺組織及NHC內(nèi)Th1/Th2型細(xì)胞因子IFN-γ、IL-5、IL-13mRNA的表達(dá)，膜表面染色檢測肺組織內(nèi)CD45+Lin-ST2+標(biāo)記的NHC數(shù)量，細(xì)胞膜內(nèi)染色檢測分泌Th1/Th2型細(xì)胞因子IFN-γ、 IL-5、IL-13的NHC數(shù)量，探討NHC在初次及再次RSV感染所誘發(fā)導(dǎo)致氣道炎癥反應(yīng)中的作用。結(jié)果顯示與再次RSV感染相比，初次RSV感染引起的肺炎癥反應(yīng)明顯加重，且肺組織內(nèi)Th2型細(xì)胞因子IL-5、IL-13mRNA表達(dá)水平亦明顯增多，提示與再次RSV感染相比，初次RSV感染可能通過誘導(dǎo)機(jī)體產(chǎn)生更多的Th2型細(xì)胞因子，進(jìn)而導(dǎo)致較重的氣道炎癥。流式細(xì)胞分析術(shù)發(fā)現(xiàn)初次RSV感染鼠肺組織內(nèi)NHC總數(shù)及IL-5+NHC，IL-13+NHC數(shù)量明顯多于再次RSV感染組，提示與再次RSV感染相比，初次RSV感染誘導(dǎo)更多的Th2型NHC進(jìn)入肺組織，參與氣道炎癥。研究證實(shí)NHC通過分泌Th2型細(xì)胞因子，尤其是IL-5和IL-13，介導(dǎo)RSV感染所誘發(fā)的氣道炎癥反應(yīng)。

呼吸道合胞病毒(RSV)；天然輔助細(xì)胞(NHC)；初次RSV感染；再次RSV感染

支氣管哮喘是由多種細(xì)胞和多種細(xì)胞因子共同參與的慢性氣道炎癥性疾病， 其主要病理改變是慢性氣道炎癥、氣道高反應(yīng)及氣道重塑[1]。病毒感染是誘發(fā)并加重哮喘的主要原因[2]。呼吸道合胞病毒(Respiratory Syncytial Virus, RSV)是呼吸道常見微生物，也是誘發(fā)和加重哮喘的主要病毒之一[3]。機(jī)體感染RSV可導(dǎo)致與哮喘發(fā)作時(shí)相似的炎癥反應(yīng)，引起氣道高反應(yīng)，從而導(dǎo)致可逆性氣道阻塞，促進(jìn)了哮喘的發(fā)展。流行病學(xué)證據(jù)顯示，呼吸道病毒感染不僅可促進(jìn)哮喘急性加重，且可造成或促進(jìn)機(jī)體對過敏原的過敏反應(yīng)，從而發(fā)展成哮喘[2]。但其中的具體作用機(jī)制尚不清楚，有研究認(rèn)為適應(yīng)性免疫細(xì)胞如CD4+T、CD8+T參與其中，但阻斷T細(xì)胞后，因RSV感染導(dǎo)致的氣道炎癥反應(yīng)依然存在[4]，故推測除T細(xì)胞以外，其他免疫細(xì)胞，特別是固有免疫細(xì)胞，可能也參與RSV感染時(shí)的氣道炎癥反應(yīng)。有文獻(xiàn)報(bào)道，固有免疫細(xì)胞如肥大細(xì)胞在RSV感染后通過STAT1途徑調(diào)控肺部嗜堿性粒細(xì)胞IL-4的表達(dá)參與[5]。也有研究認(rèn)為巨噬細(xì)胞在RSV感染階段產(chǎn)生炎性細(xì)胞因子，并且通過局部細(xì)胞因子和趨化因子的作用促進(jìn)肺部炎性癥狀[6]。有最新研究發(fā)現(xiàn)天然輔助細(xì)胞(natural helper cell，NHC)在RSV誘導(dǎo)導(dǎo)致氣道炎癥反應(yīng)中，通過IL-33/ST2途徑產(chǎn)生IL-13，導(dǎo)致嗜酸性細(xì)胞浸潤和氣道的高反應(yīng)性，加重和誘發(fā)哮喘的發(fā)生[7]。但這些研究大多是針對于初次RSV感染。NHC在初次感染及再次RSV感染所誘發(fā)的不同炎癥反應(yīng)中作用如何尚無相關(guān)報(bào)道。故在本研究中，利用初次和再次RSV感染的BALB/c鼠模型，探討NHC介導(dǎo)氣道炎性反應(yīng)的作用，從NHC這一新視角，揭示初次及再次RSV感染對哮喘發(fā)生發(fā)展的影響作用。

1 材料與方法

1.1 材料

1.1.1 動(dòng)物 4周齡雌性BALB/c鼠，SPF級動(dòng)物飼養(yǎng)室內(nèi)常規(guī)飼養(yǎng)。

1.1.2 細(xì)胞株 人喉癌上皮細(xì)胞系Hep-2為本實(shí)驗(yàn)室保存細(xì)胞株，用含10%FCS的RPMI-1640常規(guī)傳代培養(yǎng)。

1.1.3 病毒 人類呼吸道合胞病毒A2型(RSV A2)，Hep-2細(xì)胞增殖病毒，30%蔗糖超速離心法分離病毒粒子，-80 ℃冰箱保存。病毒滴度用組織細(xì)胞半數(shù)感染量(50% tissue culture infectio-ns dose，TCID50)表示。

1.1.4 試劑 RNA提取試劑盒、逆轉(zhuǎn)錄試劑盒和熒光實(shí)時(shí)定量PCR試劑盒購自TaKaRa生物工程有限公司；FITC anti-mouse lineage、PE anti-mouse McAb、APC anti-mouse CD45購自 Biolegend公司；Percp anti-Mouse ST2(IL-33R)購自 eBioscience公司。

1.2 方法

1.2.1 感染模型 4周齡雌性BALB/c鼠隨機(jī)分成初次感染組和再次感染組。初次感染組為4周齡鼠飼養(yǎng)4周后經(jīng)鼻感染2×105TCID50的RSV病毒；再次感染模型為4周齡鼠經(jīng)鼻感染2×105TCID50RSV病毒，4周后，再次經(jīng)鼻感染相同劑量RSV。二組鼠均在感染后第2天收集相關(guān)實(shí)驗(yàn)材料，用于實(shí)驗(yàn)研究。

1.2.2 肺炎性細(xì)胞分類計(jì)數(shù) 用1 mL PBS沖洗BALB/c鼠肺泡，收集肺泡灌洗液，離心后取細(xì)胞懸液做涂片，吉姆薩染色，顯微鏡下隨機(jī)選擇視野，計(jì)數(shù)200個(gè)細(xì)胞，分別計(jì)數(shù)炎性細(xì)胞如嗜酸性粒細(xì)胞和中性粒細(xì)胞數(shù)量及其百分比。

1.2.3 肺組織病理標(biāo)本制作及染色 取模型鼠左下肺，制作石蠟切片，常規(guī)HE染色，光學(xué)顯微鏡觀察肺組織形態(tài)結(jié)構(gòu)及肺炎癥反應(yīng)。

1.2.4 Real-time RT-PCR 技術(shù)檢測細(xì)胞因子mRNA的表達(dá) 利用TRIzol試劑盒提取肺組織及NH細(xì)胞的RNA, SuperscriptⅡ逆轉(zhuǎn)錄試劑盒合成cDNA。采用Real-time RT-PCR技術(shù)檢測模型鼠肺組織及肺提取的NH細(xì)胞的Th1型細(xì)胞因子IFN-γ和Th2型細(xì)胞因子IL-5、IL-13的mRNA表達(dá)水平。引物序列見表1。

表1 引物序列Table 1 The sequences of the PCR amplication primers

1.2.5 肺淋巴細(xì)胞的提取 模型鼠深度麻醉后用20 mL PBS經(jīng)心臟灌流去除血管內(nèi)淋巴細(xì)胞。取肺組織，剪碎后用200 μg/mL 膠原蛋白酶D和40 μg/mL DNaseⅠ消化1.5 h，制成單細(xì)胞懸液，利用淋巴細(xì)胞分離液分離淋巴細(xì)胞。

1.2.6 細(xì)胞膜表面染色 將肺組織淋巴細(xì)胞濃度調(diào)整至1×106個(gè)/mL。分別加入單克隆抗體(FITC-anti-mouse-lineage，PerCP-anti-mouse-ST2，APC-anti-mouse-CD45)，避光冰浴30 min， 表達(dá)CD45+Lin-ST2+的細(xì)胞為天然輔助細(xì)胞。

1.2.7 細(xì)胞內(nèi)染色 肺淋巴細(xì)胞加入PMA+ionomycin+BFA刺激培養(yǎng)5 h后，膜表面標(biāo)記CD45+Lin-ST2+的NHC，然后細(xì)胞破膜后加入PE-anti-mouse IL-5、IL-13、IFN-γ抗體，避光冰浴30 min，流式細(xì)胞儀檢測分析IL-5+的CD45+Lin-ST2+標(biāo)記的NHC、IL-13+的CD45+Lin-ST2+標(biāo)記的NHC，IFN-γ+的CD45+Lin-ST2+標(biāo)記的NHC的數(shù)量。

2 結(jié)果與分析

2.1 初次及再次RSV感染導(dǎo)致不同的肺炎癥反應(yīng) 如圖1所示，初次和再次RSV感染均導(dǎo)致BALB/c鼠肺泡灌洗液中炎性細(xì)胞數(shù)量增多。但與再次RSV感染鼠相比，初次RSV感染鼠誘導(dǎo)更多的炎性細(xì)胞，如中性粒細(xì)胞、嗜酸性粒細(xì)胞和淋巴細(xì)胞侵入肺組織。HE染色亦觀察到初次感染組肺組織炎性細(xì)胞浸潤明顯，小支氣管和周圍組織有明顯的炎癥細(xì)胞浸潤，而再次感染組炎癥反應(yīng)明顯減輕(圖2)。

圖1 BALB/c鼠感染RSV后肺泡 灌洗液中炎性細(xì)胞數(shù)量Fig.1 The number of inflammatory cells in the BAL fluids of BALB/c mice after RSV infection *P<0.05，**P<0.01 與初次感染組對比，下圖同 *P<0.05, **P<0.01, compared with the primary infection

圖2 初次RSV感染及再次感染肺病理改變Fig.2 Primary RSV infection and reinfection of pulmonary pathological changes

2.2 初次及再次感染RSV誘導(dǎo)產(chǎn)生Th1/Th2型細(xì)胞因子水平不同

Real-time RT-PCR檢測肺組織內(nèi)Th1/Th2型細(xì)胞因子mRNA的表達(dá)水平。結(jié)果顯示，對比再次RSV感染組，初次RSV感染鼠肺組織內(nèi)Th1型細(xì)胞因子IFN-γmRNA明顯減少，而Th2型細(xì)胞因子IL-5、IL-13mRNA增多，提示Th1/Th2型細(xì)胞因子平衡失調(diào)可能是導(dǎo)致氣道炎癥反應(yīng)不同的重要原因(圖3)。

圖3 RSV感染鼠肺組織內(nèi)Th1/Th2型細(xì)胞因子mRNA表達(dá)水平Fig.3 The expression of Th1/Th2 cytokine mRNA in the lungs of mice after RSV infection

2.3 NHC參與RSV感染所誘發(fā)的氣道炎癥反應(yīng)

應(yīng)用流式細(xì)胞術(shù)檢測發(fā)現(xiàn)，與再次RSV感染組相比，初次RSV感染組BALB/c鼠肺組織內(nèi)NHC數(shù)量以及分泌Th2型細(xì)胞因子IL-5、IL-13的NHC數(shù)量均明顯增多，但分泌Th1型細(xì)胞因子IFN-γ的NHC數(shù)量無明顯變化(圖4)。Real-time RT-PCR檢測技術(shù)進(jìn)一步發(fā)現(xiàn)初次感染RSV，肺組織NHC內(nèi)IL-5、IL-13 mRNA表達(dá)水平亦明顯高于再次RSV感染組鼠(圖5)，提示RSV初次感染可能通過誘導(dǎo)Th2型NH細(xì)胞肺浸潤，加重感染鼠肺局部炎癥。

圖4 肺組織NHC的絕對數(shù)量及產(chǎn)生IL-5/IL-13 NHC數(shù)量Fig.4 The absoulute number of NHC as well as IL-5/IL-13-producing NHC in the lungs

圖5 肺NHC IL-5/IL-13mRNA表達(dá)水平Fig.5 The expression of IL-5/IL-13mRNA in pulmonary NHC

3 討 論

RSV感染不能建立較長久的免疫記憶和免疫保護(hù)，故RSV反復(fù)感染的情況極為常見。但機(jī)體初次及再次感染RSV時(shí)會(huì)出現(xiàn)不同的免疫學(xué)反應(yīng)。研究發(fā)現(xiàn)，當(dāng) BALB/c鼠初次感染RSV時(shí)引發(fā)氣道高反應(yīng)性，肺泡灌洗液(BALF)中炎性細(xì)胞增多(圖1)，HE染色時(shí)肺組織病理反應(yīng)明顯(圖2)。RSV感染致氣道淋巴細(xì)胞聚集，產(chǎn)生多種細(xì)胞因子，并促使炎性介質(zhì)釋放，加速嗜酸粒細(xì)胞成熟、活化和氣道浸潤,導(dǎo)致肺出現(xiàn)炎性癥狀[8]。與初次RSV感染組相比，再次RSV感染時(shí)BALB/c鼠肺泡灌洗液中炎性細(xì)胞數(shù)量減少，且肺組織的病理反應(yīng)減輕(圖2)，提示再次RSV感染可能通過誘導(dǎo)免疫保護(hù)，從而降低氣道高反應(yīng)性，減少嗜酸粒細(xì)胞肺浸潤及氣道黏液分泌[9]。研究顯示，機(jī)體感染RSV時(shí)，表現(xiàn)為Th2型細(xì)胞因子分泌增加，誘導(dǎo)機(jī)體產(chǎn)生更多的IgE類抗體，導(dǎo)致氣道炎癥反應(yīng)加重，誘發(fā)哮喘的發(fā)生及加重[10]。Th2型細(xì)胞因子的主要代表為IL-5、IL-13，在RSV感染誘導(dǎo)的哮喘中發(fā)揮重要作用。當(dāng)機(jī)體感染RSV后，IL-5分泌增加導(dǎo)致氣道重塑及氣道上皮下纖維增生，尤其是對嗜酸性細(xì)胞的成熟、分化、激活發(fā)揮重要作用。嗜酸性細(xì)胞的增多導(dǎo)致肺浸潤加重，誘發(fā)及加重哮喘的發(fā)生[11]。而IL-13過度表達(dá)促進(jìn)氣道黏液分泌、刺激氣道上皮細(xì)胞釋放嗜酸粒細(xì)胞趨化因子，誘導(dǎo)氣道高反應(yīng)性及IgE合成，同時(shí)導(dǎo)致杯狀細(xì)胞增生和上皮的纖維化，并刺激肺成纖維細(xì)胞增殖，參與氣道重建，對哮喘的發(fā)生發(fā)展具有重要作用[12-13]。本研究顯示，初次RSV感染可誘導(dǎo)產(chǎn)生更多的Th2型細(xì)胞因子。感染鼠肺組織內(nèi)IL-5、IL-13mRNA表達(dá)水平增多，Th1型細(xì)胞因子IFN-γmRNA表達(dá)水平明顯減少。與此相反，當(dāng)再次感染發(fā)生時(shí)，肺組織內(nèi)Th2型細(xì)胞因子分泌減少，而Th1型細(xì)胞因子分泌增多(圖3)，提示再次感染可能通過提高機(jī)體Th1型應(yīng)答，下調(diào)Th2型應(yīng)答，從而減輕RSV感染所導(dǎo)致的氣道炎癥病變。本研究發(fā)現(xiàn)，與再次RSV感染組相比，初次RSV感染組誘導(dǎo)肺組織局部浸潤更多地表達(dá)CD45+Lin-ST2+的炎性細(xì)胞，即天然輔助細(xì)胞(圖4)，提示NHC可能在介導(dǎo)RSV誘發(fā)的氣道炎癥發(fā)生方面發(fā)揮重要作用。NHC屬于固有淋巴細(xì)胞家族，其區(qū)別于T、B、NK細(xì)胞[14]，可在IL-25、IL-33刺激下分泌IL-5與IL-13等Th2型細(xì)胞因子[15-16]。NHC分泌Th2型細(xì)胞因子IL-5與IL-13，進(jìn)而促進(jìn)嗜酸性粒細(xì)胞與杯狀細(xì)胞增生，引起呼吸道粘液分泌、平滑肌收縮，導(dǎo)致氣道的炎癥反應(yīng)及氣道高反應(yīng)[17-19]。在病毒誘導(dǎo)的氣道炎癥反應(yīng)中，NHC是Th2型細(xì)胞因子的重要來源[20]。有文獻(xiàn)報(bào)道，由流感病毒(IVA)感染所引起的呼吸道炎癥反應(yīng)主要是肺部NHC發(fā)揮作用。流感病毒(H3N1)感染誘導(dǎo)NHC產(chǎn)生Th2型細(xì)胞因子IL-5和IL-13，介導(dǎo)氣道高反應(yīng)性[21-22]。本研究發(fā)現(xiàn)初次感染RSV明顯增加BALB/c鼠肺淋巴細(xì)胞NHC總數(shù)，且IL-5+NHC、IL-13+NHC數(shù)量也明顯增多(圖4)。病毒感染誘導(dǎo)NHC分泌更多的Th2型細(xì)胞因子如IL-5、IL-13(圖5)，提示NHC在RSV感染導(dǎo)致氣道炎癥反應(yīng)的發(fā)生發(fā)展過程中起促進(jìn)作用。最新研究顯示，在BALB/c鼠初次感染RSV時(shí)，肺NHC產(chǎn)生大量的IL-13，誘發(fā)嗜酸細(xì)胞肺浸潤[8]，進(jìn)一步證實(shí)，NHC參與RSV感染所誘發(fā)氣道炎癥反應(yīng)。

哮喘的發(fā)生與發(fā)展與眾多因素相關(guān)。有大量研究證實(shí)呼吸道病毒感染與哮喘發(fā)作密切相關(guān)，但其在誘發(fā)哮喘中的具體作用機(jī)制尚未清楚，還需要更多的資料和實(shí)驗(yàn)來證實(shí)。本研究從固有免疫細(xì)胞天然輔助細(xì)胞的角度研究其中的作用，為提示兩者間的相關(guān)性提供進(jìn)一步的佐證。

[1] Mailaparambil B,Grychtol R,Heinzmann A.Respiratory syncytial virusbronchiolitis and asthma-insights from recent studies and implications fortherapy[J]. Inflamm Allergy Drug Targets,2009, 8(3): 202-207.

[2] Schwarze J,Gelfand EW.Respiratory viral infections as promoters ofallergic sensitization and asthma in animal models[J]. Eur Respir J, 2002 ,19(2): 341-349.

[3] Kato M, Ishioka T, Kita H, et al. Eosinophil granular proteins damagebronchial epithelial cells infected with respiratory syncytial virus[J]. Int Arch Allergy Immunol, 2012, 158 Suppl 1:11-18.

[4] Tregoning JS, Yamaguchi Y, Wang B, et al. Genetic susceptibility to thedelayed sequelae of neonatal respiratory syncytial virus infection is MHCdependent[J]. J Immunol, 2010,185(9): 5384-5391.

[5] Moore ML,Newcomb DC,Parekh VV,et al.STAT1 negatively regulateslung basophil IL-4 expression induced by respiratory syncytial virus infection[J]. J Immunol, 2009, 183(3): 2016-2026.

[6] Pribul PK, Harker J, Wang B, et al. Alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development[J]. J Virol, 2008, 82(9): 4441-4448.

[7] Jing Liu, Jianqi Wu, Feifei Qi, et al. Natural helper cells contribute topulmonary eosinophilia by producing IL-13 viaIL-33/ST2 pathway inamurine model of respiratory virus infection[J]. Int Immunopharma col, 2015, 28(1): 337-343.

[8] Papi A, Johnston SL. Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (ICAM-1) viaincreased NF-kappaB-mediated transcription[J]. J Biol Chem, 1999, 274(14): 9707-9720.

[9] Hamelmann E, Gelfand EW. Role of IL-5 inthe development of allergen indued airway hyper responsiveness [J]. Int Arch Allergy Immunol, 1999, 120(1):8-16.

[10]Kellner J, Gamarra F, Welsch U, et al. IL-13 Ralpha2 reverses the effects of IL-13 and IL-4 on bronchial reactivity and acetylcholine-induced Ca2+signaling[J]. Int Arch Allergy Immunol, 2007, 142(3): 199-210.

[11]Halim TY, Krauss RH, Sun AC, et al. Lung natural helper cells are acritical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation[J]. Immunity, 2012, 36(3): 451-463.

[12]Dakhama A, Park JW, Taube C, et al. Alteration of airway neuropeptideexpression and development of airway hyperresponsiveness following grespiratory syncytial virus infection[J]. Am J Physiol Lung Cell Mol Physiol, 2005, 288(4): 761-770.

[13]Cautivo KM, Bueno SM, Cortes CM, et al. Efficient lung recruitment ofrespiratory syncytial virus-specific Th1 cells induced by recombinant bacillus Calmette-Guérin promotes virus clearance and protects from infection[J]. J Immunol, 2010, 185(12): 7633-7645.

[14]Moro K, Yamada T, Tanabe M, et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells[J]. Nature, 2010, 463(7280): 540-544.

[15]Fort MM, Cheung J, Yen D, et al. IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo[J]. Immunity, 2001,15(6): 985-995.

[16]Barlow JL, McKenzie AN.Type-2 innate lymphoid cells in human allergicdisease[J]. Curr Opin Allergy Clin Immunol, 2014,14(5): 397-403.

[17]Ali S,Huber M,Kollewe C, et al. IL-1 receptor accessory protein isessential for IL-33-induced activation of T lymphocytes and mast cells[J]. Proc Natl Acad Sci USA, 2007, 104(47):18660-18665.

[18]Smithgall MD, Comeau MR, Yoon BR, et al. IL-33 amplifies both Th1-and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells[J]. IntImmunol, 2008, 20(8): 1019-1030.

[19]Suzukawa M, Iikura M, Koketsu R, Nagase H,et al. An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor[J].J Immuno, 2008, 181(9): 5981-5989.

[20]Gorski SA, Hahn YS, Braciale TJ.Group 2 innate lymphoid cell productionof IL-5 is regulated by NKT cells during influenza virus infection[M]. PLoSPatogh, 2013,9(9):e1003615.

[21]Chang YJ, Kim HY, Albacker LA, et al. Innate lymphoid cells mediateinfluenza-induced airway hyper-reactivity independently of adaptive immunity[J]. Nat Immunol, 2011,12(7): 631-638.

[22]Lukacs NW, Tekkanat KK, Berlin A, et al. Respiratory syncytial viruspredisposes mice to augmented allergic airway responses via IL-13-mediatedmechanisms [J]. J Immunol, 2001,167(2): 1060-1065.

The Role of Natural Helper Cells Play in Airway Inflammation Induced by Primary and Secondary RSV Infection

HU Hai-yan, XU Lei, QI Fei-fei, WANG Dan-dan, LIU Jing, ZENG Sheng, LIU Bei-xing

(Teach. &Res.Div.ofImmunol.,Schl.ofBasicMed.Sci.,ChinaMed.Uni.,Shenyang110122)

Natural helper cells (NHC) are ones of the type 2 innate lymphocytes found recently. It has been reported that NHC can secret large amounts of cytokine type Th2, therefore, they play important role during the airway inflammation induced by viral infection. RSV is the most common respiratory virus, it is also a process of repeated infection. NHC may play an important role in RSV-induced airway inflammation. However, the role of NHC play in RSV infection, particularly in secondary infection is still unclear. Therefore, in this study a model of primary and secondary infection was constructed, and the role of NHC in RSV-induced airway inflammation has been observed. By using BALB/c mice that were infected intranasally with RSV, the number of inflammatory cells in the BAL fluids was counted. Lung histopathology was determined by HE staining. The expression of IFN-γ, IL-5, IL-13 mRNA in lung tissue are determined and NHC by RT-PCR. The number of CD45+Lin-ST2+marked NHC and IL-5+NH cells, IL-13+NHC on membrane surface in lung tissue, the number of NHC of IFN-γ, IL-5, IL-13 type Th1/Th2 cytokine were determined by intracellular membrane staining, to investigate the role of NHC in RSV induced by primary and secondary infections. The results showed that as compared with the secondary RSV infection, the lung inflammation reaction caused by RSV infection was worsened significantly, moreover, the expression level of cytokine IL-5, IL-13mRNA type Th2 increased significantly, suggested that as compared with the secondary infection, the primary RSV infection might induce the organism to produce more cytokine Th2, and caused the airway inflammation to worsen. Flow cytometry analysis has found that the total number of NHC in mice lung tissue during RSV primary infection as well as the number of IL-5+NHC, IL-13+NHC were more than the secondary infection group, suggested that as compared with the secondary infection, the RSV primary infection induce more NHC of type Th2 to enter into lung tissue and take part the airway inflammation. The study has proved that NHC intermediately induce airway inflammation through secretion of cytokine of type Th2, particularly IL-5 and IL-13.

RSV; natural helper cells (NHC); RSV primary and secondary infection

國家自然科學(xué)基金項(xiàng)目(81273239/H1005 )

胡海燕 女，碩士研究生。主要從事病毒與哮喘研究。E-mail：hyhu1821@aliyun.com

* 通訊作者。女，教授，博士生導(dǎo)師。主要從事病毒與哮喘方面的研究。E-mail：bxliu@mail.cmu.edu.cn

2015-08-10；

2015-09-20

Q939.93;R392.1

A

1005-7021(2016)05-0038-06

10.3969/j.issn.1005-7021.2016.05.007